Releasable linkage and compositions containing same

ABSTRACT

A compound comprised of a hydrophilic polymer covalently yet reversibly linked to a amine-containing ligand through a dithiobenzyl linkage is described.

This application is a continuation of U.S. application Ser. No.09/982,336 filed Oct. 15, 2001, now U.S. Pat. No. 6,605,299; which is acontinuation of U.S. application Ser. No. 09/556,056 filed Apr. 21,2000, now U.S. Pat. No. 6,342,244; which claims the benefit of U.S.Provisional Application No. 60/130,897 filed Apr. 23, 1999, nowabandoned; all of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to a compound comprised of a hydrophilicpolymer, such as polyethyleneglycol, cleavably linked to anamine-containing ligand, which in preferred embodiments can be anamine-containing lipid, drug or protein. The compounds are cleavableunder mild thiolytic conditions to regenerate the amine-containingligand in its original form.

BACKGROUND OF THE INVENTION

Hydrophilic polymers, such as polyethylene glycol (PEG), have been usedfor modification of various substrates, such as polypeptides, drugs andliposomes, in order to reduce immunogenicity of the substrate and/or toimprove its blood circulation lifetime.

For example, parenterally administered proteins can be immunogenic andmay have a short pharmacological half-life. Proteins can also berelatively water insoluble. Consequently, it can be difficult to achievetherapeutically useful blood levels of the proteins in patients.Conjugation of PEG to proteins has been described as an approach toovercoming these difficulties. Davis et al. in U.S. Pat. No. 4,179,337disclose conjugating PEG to proteins such as enzymes and insulin to formPEG-protein conjugates having less immunogenicity yet which retain asubstantial proportion of physiological activity. Veronese et al.(Applied Biochem. and Biotech, 11:141-152 (1985)) disclose activatingpolyethylene glycols with phenyl chloroformates to modify a ribonucleaseand a superoxide dimutase. Katre et al. in U.S. Pat. Nos. 4,766,106 and4,917,888 disclose solubilizing proteins by polymer conjugation. PEG andother polymers are conjugated to recombinant proteins to reduceimmunogenicity and increase half-life. (Nitecki et al., U.S. Pat. No.4,902,502; Enzon, Inc., PCT/US90/02133). Garman (U.S. Pat. No.4,935,465) describes proteins modified with a water soluble polymerjoined to the protein through a reversible linking group.

However, PEG-protein conjugates described to date suffer from severaldisadvantages. For example, modification of the protein with PEG ofteninactivates the protein so that the resulting conjugate has poorbiological activity. Typically in the prior art to date, it is desiredto have the PEG stably linked to the protein so that the beneficialproperties provided by PEG remain. Another problem with some protein PEGconjugates is that upon decomposition of the conjugate undesirableproducts may be formed.

PEG has also been described for use in improving the blood circulationlifetime of liposomes (U.S. Pat. No. 5,103,556). Here, the PEG iscovalently attached to the polar head group of a lipid in order to maskor shield the liposomes from being recognized and removed by thereticuloendothelial system. Liposomes having releasable PEG chains havealso been described, where the PEG chain is released from the liposomeupon exposure to a suitable stimulus, such as a change in pH(PCT/US97/18813). However, release of the PEG chain from the liposomesuffers from the drawback that the decomposition products are chemicallymodified and can have unpredictable, potentially negative effects invivo.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a compoundwhere a ligand is covalently yet reversibly linked to a hydrophilicpolymer. Upon cleavage of the linkage, the ligand in its native form isregenerated.

In one aspect, the invention includes a compound having the generalstructure:

wherein R¹ is a hydrophilic polymer comprising a linkage for attachmentto the dithiobenzyl moiety; R² is selected from the group consisting ofH, alkyl and aryl; R³ is selected from the group consisting of O(C═O)R⁴,S(C═O)R⁴, and O(C═S)R⁴; R⁴ comprises an amine-containing ligand; and R⁵is selected from the group consisting of H, alkyl and aryl; and whereorientation of CH₂—R³ is selected from the ortho position and the paraposition.

In one embodiment, R⁵ is H and R² is selected from the group consistingof CH₃, C₂H₅ and C₃H₈. In another embodiment, R² and R⁵ are alkyls.

In another embodiment, the amine-containing ligand R⁴ is selected fromthe group consisting of a polypeptide, an amine-containing drug and anamine-containing lipid. In an embodiment where the amine-containingligand R⁴ is an amine-containing lipid, the lipid includes either asingle hydrocarbon tail or a double hydrocarbon tail. In one preferredembodiment, the lipid is a phospholipid having a double hydrocarbontail.

The hydrophilic polymer R¹ can be, in yet another embodiment, selectedfrom the group consisting of polyvinylpyrrolidone, polyvinylmethylether,polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropyl-methacrylamide, polymethacrylamide,polydimethyl-acrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide, copolymers thereof, andpolyethyleneoxide-polypropylene oxide.

In one preferred embodiment, the hydrophilic polymer R¹ ispolyethyleneglycol. In another embodiment, when R¹ is polyethyleneglycol, R⁵ is H and R² is CH₃ or C₂H₅.

In still another embodiment, the amine-containing ligand R⁴ is apolypeptide. The polypeptide can be, in another embodiment, arecombinant polypeptide. Exemplary and preferred polypeptides includecytokines, such as interferons, interleukins, and growth factors, andenzymes.

In another aspect, the invention includes a composition comprising aconjugate obtainable by reaction with a compound having the generalstructural formula:

wherein R¹ is a hydrophilic polymer comprising a linkage for attachmentto the dithiobenzyl moiety; R² is selected from the group consisting ofH, alkyl and aryl; R³ is selected from the group consisting of O(C═O)R⁴,S(C═O)R⁴, and O(C═S)R⁴; R⁴ comprises a leaving group; and R⁵ is selectedfrom the group consisting of H, alkyl and aryl; and where orientation ofCH₂—R³ is selected from the ortho position and the para position. Thecomposition also includes a pharmaceutically-acceptable carrier, such assaline, buffer or the like.

In one embodiment of this aspect, R² is selected from the groupconsisting of CH₃, C₂H₅ and C₃H₈.

In another embodiment, R³ is O(C═O)R⁴ and R⁴ is a hydroxy- oroxy-containing leaving group. The leaving group, in another embodiment,is derived from a compound selected from the group consisting ofchloride, para-nitrophenol, ortho-nitrophenol,N-hydroxy-tetrahydrophthalimide, N-hydroxysuccinimide,N-hydroxy-glutarimide, N-hydroxynorbornene-2,3-dicarboxyimide,1-hydroxybenzotriazole, 3-hydroxypyridine, 4-hydroxypyridine,2-hydroxypyridine, 1-hydroxy-6-trifluoromethylbenzotriazole, imidazole,triazole, N-methyl-imidazole, pentafluorophenol, trifluorophenol andtrichlorophenol.

In one embodiment, the claimed compound is reacted with anamine-containing ligand that displaces R⁴ to form a conjugate thatincludes the amine-containing ligand. For example, the amine-containingligand can be a phospholipid.

In a preferred embodiment, the hydrophilic polymer R¹ ispolyethyleneglycol, R⁵ is H and R² is CH₃ or C₂H₅.

In yet another aspect of this embodiment, the composition containing theconjugate comprises a liposome. The liposome can further comprise anentrapped therapeutic agent.

In another embodiment, the amine-containing ligand comprises apolypeptide.

In yet another aspect, the invention includes a liposome compositioncomprising liposomes which include a surface coating of hydrophilicpolymer chains wherein at least a portion of the hydrophilic polymerchains have the general structure:

wherein R¹ is a hydrophilic polymer comprising a linkage for attachmentto the dithiobenzyl moiety; R² is selected from the group consisting ofH, alkyl and aryl; R³ is selected from the group consisting of O(C═O)R⁴,S(C═O)R⁴, and O(C═S)R⁴; R⁴ comprises an amine-containing ligand; and R⁵is selected from the group consisting of H, alkyl and aryl; and whereorientation of CH₂—R³ is selected from the ortho position and the paraposition. The liposomes have a longer blood circulation lifetime thanliposomes having hydrophilic polymer chains joined to the liposome viaan aliphatic disulfide linkage.

In one embodiment, the liposome further comprises an entrappedtherapeutic agent.

In still another aspect, the invention includes a method for improvingthe blood circulation lifetime of liposomes having a surface coating ofreleasable hydrophilic polymer chains. The method includes preparingliposomes that have between about 1% to about 20% of a compound havingthe general structure:

wherein R¹, R², R³, and R⁵ are as described above and R⁴ comprises anamine-containing lipid.

In a preferred embodiment of this aspect, R⁵ is H and R² is selectedfrom the group consisting of CH₃, C₂H₅ and C₃H₈.

In another embodiment, the amine-containing lipid comprises aphospholipid and R¹ is polyethyleneglycol.

In this aspect, the liposomes can further comprise an entrappedtherapeutic agent.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an embodiment of the invention where the dithiobenzyl(DTB) links a methoxy-polyethyelene glycol (mPEG) moiety and theamine-containing ligand;

FIG. 1B shows the products after thiolytic cleavage of the compound inFIG. 1A;

FIG. 2 illustrates a synthetic reaction scheme for synthesis of themPEG-DTB-amine-lipid, where the amine-ligand is the lipiddistearoylphosphatidylethanolamine (DSPE);

FIG. 3 illustrates the thiolytic cleavage mechanism of apara-dithiobenzyl urethane (DTB)-linked mPEG-DSPE conjugate;

FIGS. 4A-4B show a synthetic reaction scheme for preparation of anmPEG-DTB-DSPE compound in accord with the invention where the DTBlinkage is sterically hindered by an alkyl group;

FIG. 5 shows another synthetic reaction scheme for preparation of anmPEG-DTB-ligand compound in accord with the invention;

FIG. 6A is a synthetic reaction scheme for synthesis of anmPEG-DTB-lipid which upon thiolytic cleavage yields a cationic lipid;

FIG. 6B shows the products after thiolytic cleavage of the compound inFIG. 6A;

FIG. 7A shows the rate of cleavage of ortho-mPEG-DTB-DSPE andpara-mPEG-DTB-DSPE conjugates in solution to form micelles in bufferalone (ortho-conjugate (*); para-conjugate (+)) and in the presence of150 μM cysteine (ortho-conjugate (open circles); para-conjugate (opensquares);

FIG. 7B shows the rate of cleavage of micellar mPEG-DTB-DSPE conjugatesas described in FIG. 7A and of ortho-mPEG-DTB-DSPE (solid circles) andpara-mPEG-DTB-DSPE (solid squares) conjugates formulated in liposomesand incubated in the presence of 150 μM cysteine;

FIGS. 8A-8B show percentage of content release of entrapped fluorophorefrom liposomes comprised of DOPE:ortho-mPEG-DTB-DSPE (FIG. 8A) or ofDOPE:para-mPEG-DTB-DSPE (FIG. 8B) incubated in the presence of cysteineat the indicated concentrations;

FIG. 9A shows normalized percent release of entrapped fluorophore as afunction of time for liposomes comprised of DOPE and para-mPEG-DTB-DSPE.The percent release of entrapped fluorophore is normalized with respectto percent release of fluorophore from liposomes incubated in theabsence of cysteine. The release rate from liposomes incubated in thepresence of cysteine at concentrations of 15 μM (solid squares), 75 μM(open triangles), 150 μM (X symbols), 300 μM (open circles), 1500 μM(solid circles), 3000 μM (+ symbols), and 15000 μM (open diamonds) isshown;

FIG. 9B shows normalized percent release of entrapped fluorophore as afunction of time for liposomes comprised of DOPE andpara-mPEG-MeDTB-DSPE. The percent release of entrapped fluorophore isnormalized with respect to percent release of fluorophore from liposomesincubated in the absence of cysteine. The release rate for liposomesincubated in the presence of cysteine at concentrations of 15 μM (solidsquares), 75 μM (open triangles), 150 μM (X symbols), 300 μM (opencircles), 1500 μM (solid circles), 3000 μM (+ symbols), and 15000 μM(open diamonds) is shown;

FIG. 9C shows normalized percent release of entrapped fluorophore as afunction of time for liposomes comprised of DOPE andmPEG-MeDTB-distearoyl-glycerol compound of FIG. 6A. The percent releaseof entrapped fluorophore is normalized with respect to percent releaseof fluorophore from liposomes incubated in the absence of cysteine. Therelease rate of dye upon cleavage of the compound from liposomesincubated in the presence of cysteine at concentrations of 15 μM (solidsquares), 75 μM (open triangles), 150 μM (X symbols), 300 μM (opencircles), 1500 μM (solid circles), 3,000 μM (+ symbols), and 15,000 μM(open diamonds) is shown;

FIG. 10 is a plot showing the amount of liposomes, in counts perminute/mL of liposomes containing entrapped In¹¹¹, in blood samplestaken from mice at various times after injection of liposomes comprisedof PHPC:cholesterol:mPEG-DTB-DSPE (55:40:5 molar ratio). One group ofanimals received a 200 μL injection of 200 mM cysteine at time zero(solid squares). The control group was injection with saline at timezero (open circles);

FIG. 11A shows a synthetic reaction scheme for synthesis of anmPEG-DTB-protein compound in accord with another embodiment of theinvention;

FIG. 11B shows the decomposition products after thiolytic cleavage ofthe compound in FIG. 11A;

FIG. 12 is a rendering of a photograph of an sodium-dodecyl-sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) profile of lysozymereacted for 15 minutes (Lane 1) or for 1 hour (Lane 2) withmPEG-MeDTB-nitrophyenylchloroformate to form a mPEG-MeDTB-lysozymeconjugate, native lysozyme (Lane 3), lysozyme reacted for 1 hour withmPEG-nitrophenylchloroformate (Lane 4), molecular weight markers (Lane5), and the samples of Lanes 1-4 treated with 2% β-mercaptoethanol for10 minutes at 70° C. (Lanes 6-9);

FIG. 13 shows the decomposition products after thiolytic cleavage of thean mPEG-DTB-p-nitroanilide conjugate;

FIG. 14A shows the absorbence as a function of wavelength, in nm, ofmPEG-MeDTB-para-nitroanilide (closed diamonds) and after in vitroincubation with 5 mM cysteine for 2 minutes (closed squares), 5 minutes(x symbols), 10 minutes (open squares), 20 minutes (triangles), 40minutes (open diamonds) and 80 minutes (closed circles); and

FIG. 14B shows the amount of para-nitroanilide, in mole/L, released invitro as a function of time, in minutes, frommPEG-MeDTB-para-nitroanilide conjugate incubated in the presence of 5 mMcysteine (closed circles), 1 mM cysteine (closed squares) and 0.15 mMcysteine (closed diamonds).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Polypeptide” as used herein refers to a polymer of amino acids and doesnot refer to a specific length of a polymer of amino acids. Thus, forexample, the terms peptide, oligopeptide, protein, and enzyme areincluded within the definition of polypeptide. This term also includespost-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations, and the like.

“Amine-containing” intends any compound having a moiety derived fromammonia by replacing one or two of the hydrogen atoms by alkyl or arylgroups to yield general structures RNH₂ (primary amines) and R₂NH(secondary amines), where R is any hydrocarbyl group.

“Hydrophilic polymer” as used herein refers to a polymer having moietiessoluble in water, which lend to the polymer some degree of watersolubility at room temperature. Exemplary hydrophilic polymers includepolyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline,polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropyl-methacrylamide, polymethacrylamide,polydimethyl-acrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide, copolymers of the above-recitedpolymers, and polyethyleneoxide-polypropylene oxide copolymers.Properties and reactions with many of these polymers are described inU.S. Pat. Nos. 5,395,619 and 5,631,018.

“Polymer comprising a reactive functional group” or “polymer comprisinga linkage for attachment” refers to a polymer that has been modified,typically but not necessarily, at a terminal end moiety for reactionwith another compound to form a covalent linkage. Reaction schemes tofunctionalize a polymer to have such a reactive functional group ofmoiety are readily determined by those of skill in the art and/or havebeen described, for example in U.S. Pat. No. 5,613,018 or by Zalipsky etal., in for example, Eur. Polymer. J., 19(12):1177-1183 (1983); Bioconj.Chem., 4(4):296-299 (1993).

“Recombinant” as in “recombinant polypeptide” implies joining of aminoacids through laboratory manipulation into a desired sequence.

“Alkyl” as used herein intends a group derived from an alkane by removalof a hydrogen atom from any carbon atom: “C_(n)H_(2n+1)”. The groupsderived by removal of a hydrogen atom from a terminal carbon atom ofunbranched alkanes form a subclass of normal alkyl (n-alkyl) groups:H[CH₂]_(n). The groups RCH₂—, R₂CH— (R not equal to H), and R₃C— (R notequal to H) are primary, secondary and tertiary alkyl groupsrespectively.

“Aryl” refers to a substituted or unsubstituted monovalent aromaticradical having a single ring (e.g., benzene) or two condensed rings(e.g., naphthyl). This term includes heteroaryl groups, which arearomatic ring groups having one or more nitrogen, oxygen, or sulfuratoms in the ring, such as furyl, pyrrole, pyridyl, and indole. By“substituted” is meant that one or more ring hydrogens in the aryl groupis replaced with a halide such as fluorine, chlorine, or bromine; with alower alkyl group containing one or two carbon atoms; nitro, amino,methylamino, dimethylamino, methoxy, halomethoxy, halomethyl, orhaloethyl.

An “aliphatic disulfide” linkage intends a linkage of the formR′—S—S—R″, where R′ and R″ are linear or branched alkyl chains that maybe further substituted.

The following abbreviations are used herein: PEG, poly(ethylene glycol);mPEG, methoxy-PEG; DTB, dithiobenzyl; MeDTB, methyl-dithiobenzyl, EtDTB,ethyl-dithiobenzyl; DSPE, distearoyl phosphatidylethanolamine; DOPE,dioleoyl phosphatidylethanolamine; PHPC, partially hydrogenatedphosphatidylcholine; MALDI-TOFMS, matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry.

II. The Compound of the Invention

In one aspect, the invention comprises a compound of the form:

wherein R¹ comprises a hydrophilic polymer including functional groupsuitable for covalently attaching the polymer to the dithiobenzylmoiety. R² and R⁵ are independently selected to be H, an alkyl or anawl, and, as will be seen, can be varied to tailor the rate of disulfidecleavage. For example, to achieve a faster rate of cleavage, R² and R⁵are hydrogen. A slower rate of cleavage is achieved by stericallyhindering the disulfide by selecting an alkyl or awl for one or both ofR² and R⁵. R³ comprises a linking moiety joined to R⁴, which comprisesan amine-containing ligand. The linking moiety in preferred embodimentsis O(C═O), S(C═O) or O(C═S). The amine-containing ligand R⁴ can be aprimary or a secondary amine and can be selected from any number ofsubstrates, including, but not limited to lipids, drugs, polypeptides,viruses, surfaces of biomaterials and aminoglycosides. In preferredembodiments, R⁴ is a primary or secondary amine-containing lipid, drugor polypeptide. In the compound of the invention, the orientation of thegroup CH₂—R³ can be either ortho or para.

FIG. 1A shows the structure of an exemplary compound in accord with theinvention, where R¹ is the hydrophilic polymer methoxy-polyethyleneglycol, mPEG=CH₃O(CH₂CH₂O)_(n) where n is from about 10 to about 2300,which corresponds to molecular weights of about 440 Daltons to about100,000 Daltons. The molecular weight of the polymer depends to someextent on the selection of R³. In embodiments where R³ is anamine-containing lipid for use in a liposome a preferred range of PEGmolecular weight is from about 750 to about 10,000 Daltons, morepreferably from about 2,000 to about 5,000 Daltons. The mPEG in thisembodiment includes a urethane linking moiety. In embodiments where R³is an amine-containing polypeptide a preferred range of PEG molecularweight is from about 2,000 to about 40,000 Daltons, more preferably fromabout 2,000 to about 20,000 Daltons. It will be appreciated that R¹ canbe selected from a variety of hydrophilic polymers, and exemplarpolymers are recited above. It will also be appreciated that for someligands, such as polypeptides, the molecular weight of the polymer maydepend on the number of polymer chains attached to the ligand, where alarger molecular weight polymer is often selected when the number ofattached polymer chains is small.

With continuing reference to FIG. 1 a, R² and R⁵ in this exemplarycompound are H, however either or both R² and R⁵ can also be a straightchain or branched alkyl or an aryl group. In a preferred embodiment, R⁵is H and R² is an alkyl, and several examples are given below. In thecompound shown in FIG. 1A, R³ takes the general form ofO(C═O)—(NH₂-ligand), where the NH₂-ligand can be any amine-containingpolypeptide, drug or lipid, and specific examples of each embodiment aregiven below. R3 can also be of the form O(C═S)—(NH₂-ligand) orS(C═O)—(NH₂-ligand).

FIG. 1B shows the mechanism of thiolytic cleavage of themPEG-DTB-(NH₂-ligand) compound of FIG. 1A. The ortho- orpara-dithiobenzyl carbamate moiety is cleavable under mild thiolyticconditions, such as in the presence of cysteine or othernaturally-occurring reducing agents. Upon cleavage, the amine-containingligand is regenerated in its natural, unmodified form. Studies insupport of the invention, described below, show that natural,physiologic conditions in vivo are sufficient to initiate and achievecleavage of the DTB linkage. It will be appreciated that a reducingagent can also be administered to artificially induce thiolyticconditions sufficient for cleavage and decomposition of the compound.

As noted above, R³ takes the general form of a linking moiety, such asO(C═O), S(C═O) or O(C═S) joined to an amine-containing ligand. Inpreferred embodiment, the amine-containing ligand comprises anamine-containing polypeptide, drug or lipid. Examples of theseembodiments will now be described.

A. Amine-Containing Lipid

In one embodiment, the amine-containing ligand is an amine-containinglipid. Lipids as referred to herein intend water-insoluble moleculeshaving at least one acyl chain containing at least about eight carbonatoms, more preferably an acyl chain containing between about 8-24carbon atoms. A preferred lipid is a lipid having an amine-containingpolar head group and an acyl chain. Exemplary lipids are phospholipidshaving a single acyl chain, such as stearoylamine, or two acyl chains.Preferred phospholipids with an amine-containing head group includephosphatidylethanolamine and phosphatidylserine. The lipid tail(s) canhave between about 12 to about 24 carbon atoms and can be fullysaturated or unsaturated. One preferred lipid isdistearoylphosphatidylethanolamine (DSPE), however those of skill in theart will appreciate the wide variety of lipids that fall within thisdescription. It will also be appreciated that the lipid can naturallyinclude an amine group or can be derivatized to include an amine group.Other lipid moieties that do not have an acyl tail, such ascholesterolamine, are also suitable.

The synthesis of a polymer-DTB-lipid compound is schematically depictedin FIG. 2. mPEG derivatives (MW 2000 and 5000 Daltons) having amethoxycarbonyldithioalkyl end group were prepared by reacting2-(methoxycarbonyldithio)ethaneamine with mPEG-chloroformate, which wasreadily prepared by phosgenation of dried mPEG-OH solution (Zalipsky,S., et al., Biotechnol. Appl. Biochem. 15:100-114 (1992).). The formercompound was obtained through 2-aminoethanethiol hydrochloride reactionwith an equivalent amount of methoxycarbonylsulfenyl chloride, accordingto published procedures (Brois, S. J., et al., J. Amer. Chem. Soc.92:7629-7631 (1970); Koneko, T., et al., Bioconjugate Chem. 2:133-141(1991)). Both the para and ortho isomers of mercaptobenzyl alcohol(Grice, R., et al., J. Chem. Soc. 1947-1954 (1963)) coupled cleanly withthe resulting PEG-linked acyldisulfide, yielding mPEG bearing a dithiobenzyl alcohol end group. Active carbonate introduction proceeded aswith underivatized mPEG-OH, to give the para-nitrophenyl carbonate.Addition of DSPE in ethanolamine formed the desired mPEG-DTB-DSPEproduct. Both ortho- and para-DTB-lipid compounds were prepared andpurified by silica gel chromatography and characterized by NMR andMALDI-TOFMS, the details of which are given in Example 1.

FIG. 3 shows the mechanism of thiolytic cleavage of the mPEG-DTB-DSPEconjugate. Upon cleavage, the phosphatidylethanolamine lipid isregenerated in its natural, unmodified form.

FIGS. 4A-4B show a reaction scheme for synthesis of mPEG-DTB-DSPEconjugates having an alkyl group adjacent the disulfide linkage, e.g., amore hindered disulfide linkage. As described more fully in Example 2A,mPEG-OH in dichloromethane was reacted with p-nitrophenylchloroformatein the presence of triethylamine (TEA) to form mPEG-nitrophenylcarbonate. An amino alcohol, such as 1-amino-2-propanol or1-amino-2-butanol, in dimethylformamide (DMF) was reacted with themPEG-nitrophenyl carbonate in the presence of TEA to form a secondaryalcohol attached to PEG. The secondary alcohol was then converted to thedesired mPEG-DTB-DSPE compound as illustrated in FIG. 4A and detailed inExample 2A.

In this reaction scheme, mPEG-methyl-dithiobenzyl-nitrophenylchloroformate was reacted with DSPE to form the desired compound. Thenitrophenyl chloroformate moiety in themPEG-methyl-dithiobenzyl-nitrophenyl chloroformate compound acts as aleaving group to yield the desired product upon reaction with a selectedlipid. The invention contemplates, in another aspect, a composition thatcomprises a compound produced by reaction with a compound such asmPEG-methyl-dithiobenzyl-R³, where R³ represents a leaving group joinedthrough a linking moiety to the benzene ring. The leaving group isdisplaced upon reaction with an amine-containing ligand, such as DSPE, apolypeptide or an amine-containing drug. The leaving group is selectedaccording to the reactivity of the amine in the ligand, and ispreferably derived from various acidic alcohols that have a hydroxy- oroxy-containing leaving group. These include chloride, p-nitrophenol,o-nitrophenol, N-hydroxy-tetrahydrophthalimide, N-hydroxysuccinimide,N-hydroxy-glutarimide, N-hydroxynorbornene-2,3-dicarboxyimide,1-hydroxybenzotriazole, 3-hydroxypyridine, 4-hydroxypyridine,2-hydroxypyridine, 1-hydroxy-6-trifluoromethylbenzotriazole, imidazole,triazole, N-methyl-imidazole, pentafluorophenol, trifluorophenol andtrichlorophenol.

Example 2B describes preparation of an mPEG-EtDTB-lipid conjugate wherethe disulfide linkage is hindered by an ethyl moiety.

FIG. 5 shows another synthetic reaction scheme for preparation of anmPEG-DTB-ligand compound in accord with the invention. The details ofthe reaction procedure are given in Examples 3A-3B. Briefly, cold1-amino-2-propanol was reacted with sulfuric acid to form2-amino-1-methylethyl hydrogen sulfate. This product was reacted withcarbon disulfide and sodium hydroxide in aqueous ethanol to yield5-methylthiazolidine-2-thione. An aqueous solution of hydrochloric acidwas added to the 5-methylthiazolidine-2-thione and heated. Afterrefluxing for one week, the product, 1-mercapto(methyl)ethyl ammoniumchloride, was crystallized and recovered. This product was reacted withmethoxy carbonylsulfenyl chloride to yield2-(methoxycarbonyldithio)ethaneamine. Reaction of the2-(methoxycarbonyldithio)ethaneamine with mPEG-chloroformate using theprocedure described above with respect to FIG. 2 yields the desiredmPEG-DTB-nitrophenyl compound suitable for reaction with a selectedamine-containing ligand to form a compound in accord with the invention.

Example 3B describes the reaction for synthesis ofmPEG-(ethyl)DTB-nitrophenyl.

FIG. 6A shows a reaction scheme for preparation of anothermPEG-DTB-lipid compound in accord with the invention. The reactiondetails are provided in Example 4. The lipid 1,2-distearoyl-sn-glycerolis activated for reaction with mPEG-DTB-nitrophenyl, prepared asdescribed in FIG. 4A or FIG. 5. The resulting mPEG-DTB-lipid differsfrom the compounds described above in the absence of a phosphate headgroup. The mPEG-DTB-lipid of FIG. 6A is neutral prior to cleavage. Asshown in FIG. 6B, upon thiolytic reduction of the disulfide bond, thecompound decomposes to yield a cationic lipid. The positively-chargedlipid provides for electrostatic interaction in vivo and commensurateadvantages in in vivo targeting.

In the reaction schemes described above, R⁵ of the claimed compound isH. However, in other embodiments R⁵ is an alkyl or an aryl moiety. Inthis approach, for example where R² and R⁵ are both CH₃ moieties, anα,β-unsaturated acyl chloride (R′R″C═CHCOCl, where R′ is, for exampleCH₃ and R″ is CH₃, however any alkyl or aryl is contemplated) is reactedwith an amine-terminated PEG to give the corresponding N-PEG-substitutedα,β-unsaturated amide. This compound is reacted with thiolacetic acid,giving the corresponding N-PEG-substituted β-(acetylthio)amide viaconjugate addition to the C═C bond. The acetylthio group (—SCOCH₃) ishydrolyzed to a thiol group (—SH), which is then reacted withmethyl(chlorosulfenyl)formate (ClSCOOCH₃), generating a methoxycarbonyldithio group (—SSCOOCH₃); this intermediate is then reacted withp-mercapto benzyl alcohol to give the N-PEG-substituted β-(dithiobenzylalcohol)amide (having the structurePEG-NH—CO—CH₂CR′R″—SS-p-phenyl-CH₂OH). The benzyl alcohol moiety is thenreacted with nitrophenyl chloroformate to give the nitrophenyl carbonateleaving group, as above.

1. In Vitro Cleavage of mPEG-DTB-DSPE Compound

The in vitro rate of cleavage of ortho-mPEG-DTB-DSPE andpara-mPEG-DTB-DSPE (prepared as described in Example 1) was studied bypreparing micellar solutions of the compounds in a buffered aqueoussolution (pH 7.2). Thiolytic cleavage of the compounds was monitored inthe presence and absence of 150 μM cysteine by analyzing fordisappearance of the compounds by HPLC, as described in Example 5. Theresults are illustrated in FIG. 7A where the ortho- and para-compoundsin the absence of cysteine (* symbols and + symbols, respectively) showno cleavage and are stable under these conditions in the absence ofcysteine. The ortho- and para-compounds, represented by the open circlesand the open squares, respectively, in the presence of 150 μM cysteinecleave as shown in FIG. 7A. The ortho-compound exhibited a slightlyfaster rate of decomposition than its para counterpart (T_(1/2)≈12minutes vs. ≈18 minutes).

2. Liposome Compositions Comprising an mPEG-DTB-Lipid Compound

a). In Vitro Characterization

In one embodiment, the mPEG-DTB-lipid compound is formulated intoliposomes. Liposomes are closed lipid vesicles used for a variety oftherapeutic purposes, and in particular, for carrying therapeutic agentsto a target region or cell by systemic administration of liposomes. Inparticular, liposomes having a surface coating of hydrophilic polymerchains, such as polyethylene glycol (PEG), are desirable as drugcarries, since these liposomes offer an extended blood circulationlifetime over liposomes lacking the polymer coating. The polymer chainsin the polymer coating shield the liposomes and form a “stiff brush” ofwater solvated polymer chains about the liposomes. Thus, the polymeracts as a barrier to blood proteins, preventing binding of the proteinand recognition of the liposomes for uptake and removal by macrophagesand other cells of the reticuloendothelial system.

Typically, liposomes having a surface coating of polymer chains areprepared by including in the lipid mixture between about 1 to about 20mole percent of the lipid derivatized with the polymer. The actualamount of polymer derivatized lipid can be higher or lower depending onthe molecular weight of the polymer. In the present invention, liposomesare prepared by adding between about 1 to about 20 mole percent of thepolymer-DTB-lipid conjugate to other liposome lipid bilayer components.As will be demonstrated in the studies described below, liposomescontaining the polymer-DTB-lipid conjugate of the invention have a bloodcirculation lifetime the is longer than liposomes containing apolymer-lipid conjugate where the polymer and lipid are joined by acleavable aliphatic disulfide bond.

In studies performed in support of the invention, liposomes comprised ofthe vesicle-forming lipid partially hydrogenated phosphatidyl cholinealong with cholesterol and the ortho-mPEG-DTB-DSPE or thepara-mPEG-DTB-DSPE compound were prepared as described in Example 6.Cysteine-mediated cleavage of the mPEG-DTB-DSPE compounds was monitoredin the presence and absence of 150 μM cysteine in an aqueous buffer. Theresults are shown in FIG. 7B, which includes the data of FIG. 7A forcomparison. In FIG. 7B, the ortho- and para-compounds in micellar formin the absence of cysteine (* symbols and + symbols, respectively) showno cleavage, which indicates stability of the conjugate in the absenceof thiols. The open circles and the open squares correspond to theortho- and para-compounds, respectively, in micellar form in thepresence of cysteine, as discussed above with respect to FIG. 7A. Thesolid circles and the solid squares correspond to the ortho- andpara-compounds, respectively, in liposomal form in the presence ofcysteine.

The data in FIG. 7B shows that both the ortho and para compound wereslightly more resistant to thiolytic cleavage when incorporated intoliposomes. Examination of the thiolysis reaction products by TLC (silicagel G, chloroform/methanol/water 90:18:2) (Dittmer, J. C., et al., J.Lipid Res. 5:126-127 (1964)) showed DSPE as the sole lipid component andanother spot corresponding to a thiol-bearing, lipid-free mPEGderivative.

In another study performed in support of the invention, liposomes wereprepared from the lipid dioleoyl phosphatidylethanolamine (DOPE) andeither the ortho-mPEG-DTB-DSPE or the para-mPEG-DTB-DSPE compound wereprepared. DOPE is a hexagonal phase lipid which alone does not formlipid vesicles. However, liposomes will form when DOPE is combined witha few mole percent of the mPEG-DTB-DSPE compound. Cleavage of themPEG-DTB-DSPE compound triggers decomposition of the liposomes andrelease of liposomally-entrapped contents. Thus, the content releasecharacteristics of such liposomes provides for a convenient quantitativeevaluation of cleavable PEG-bearing liposomes.

Liposomes comprised of DOPE and the ortho- or para-mPEG-DTB-DSPEcompound were prepared as described in Example 7A with entrappedfluorophores, p-xylene-bis-pyridinium bromide and trisodium8-hydroxypyrenetrisulfonate. Release of the fluorophores from liposomesincubated in the presence of cysteine at various concentrations wasmonitored as described in Example 7B.

Results for liposomes comprising the ortho-compound are shown in FIG.8A, where percentage of content release of entrapped fluorophore fromliposomes incubated in the presence of cysteine at concentrations of 15μM (solid diamonds), 150 μM (solid inverted triangles), 300 μM (solidtriangles) and 1.5 mM (solid circles) are shown. FIG. 8B is a similarplot for liposomes comprising the para-compound, where the liposomes areincubated in cysteine at concentrations of 15 μM (solid diamonds), 300μM (solid triangles), 1 μM (solid squares) and 1.5 mM (solid circles).

FIGS. 8A-8B show that both the ortho- and para-compounds whenincorporated into liposome are cleaved, as evidenced by release of theentrapped dye, at a rate dependent on the concentration of cysteine.Control studies with non-cleavable mPEG-DSPE containing liposomesproduced no content release (results not shown here). These results alsosuggest that the ortho conjugate is somewhat more susceptible tothiolytic cleavage. For example, 300 μM cysteine liberates most of thecontents of DOPE liposomes within 20 minutes. Under the same conditions,only a fraction of liposomes having para-mPEG-DTB-DSPE decomposed.Similarly, after incubation for 20 minutes at 150 μM cysteine, half ofthe entrapped contents was released for the ortho-containing liposomes,while only approximately 10% of the contents were release in liposomescontaining the para-compound. Both ortho and para compounds havehalf-lives of less than 20 minutes at a cysteine level of 150 μM (seedata in FIG. 7B). This suggests that more than half of the originalthree mole percent of the mPEG-DTB-lipid must be cleaved to observecontent release from the liposomes.

Decomposition of the mPEG-DTB-DSPE/DOPE liposomes in 15 μM cysteine, theaverage plasma concentration in both humans and rodents (Lash, L. H., etal., Arch. Biochem. Biophys. 240:583-592 (1985)), was minimal in thetime frame of these experiments (60 minutes). This suggests that themPEG-DTB-lipid compounds should have sufficiently long lifetimes inplasma to allow the PEG-grafted vesicles to distribute systemically invivo, or to accumulate in a specific site either passively or throughligand-mediated targeting. Local or short term increase in cysteineconcentration can potentially be achieved by its intravenous orintra-arterial administration. The results shown in FIGS. 8A-8B alsosuggest that a prolonged exposure to the natural plasma cysteineconcentration (≈15 μM) would be sufficient to decompose most of thesecompounds. These suggestions were studied in in vivo experiments,described below.

In another study performed in support of the invention, liposomescomprised of DOPE and three different mPEG-DTB-lipid compounds wereprepared. The liposomes were prepared as described in Example 7 andincluded and entrapped fluorophore. The three mPEG-DTB-lipid compoundswere mPEG-DTB-DSPE as shown in FIG. 1A; mPEG-MeDTB-DSPE as shown in FIG.4B, where R is CH₃, and mPEG-MeDTB-distearoyl-glycerol, as shown in FIG.6A. The liposomes were comprised of 97 mole percent DOPE and 3 molepercent of one of the mPEG-DTB-lipid compounds. Cysteine-mediated rateof cleavage of the compounds was determined by monitoring the release ofentrapped fluorophore as a function of time in the presence of variouscysteine concentrations. The results are shown in FIGS. 9A-9C where thepercent release of entrapped fluorophore is normalized for the releaserate from liposomes incubated in buffer alone.

FIG. 9A shows the percent release of entrapped fluorophore as a functionof time for liposomes comprised of DOPE and para-mPEG-DTB-DSPE (compoundof FIG. 1A). The release rate from liposomes containing the conjugateand incubated in the presence of cysteine at concentrations of 15 μM(solid squares), 75 μM (open triangles), 150 μM (X symbols), 300 μM(open circles), 1500 μM (solid circles), 3000 μM (+ symbols), and 15000μM (open diamonds) is shown.

FIG. 9B shows the percent release of entrapped fluorophore as a functionof time for liposomes comprised of DOPE and para mPEG-MeDTB-DSPE(compound of FIG. 4B). The release rate of the fluorophore fromliposomes incubated in the presence of cysteine at concentrations of 15μM (solid squares), 75 μM (open triangles), 150 μM (X symbols), 300 μM(open circles), 1500 μM (solid circles), 3000 μM (+ symbols), and 15000μM (open diamonds) is shown.

FIG. 9C is a similar plot for liposomes formed with DOPE andmPEG-MeDTB-distearoyl glycerol (compound of FIG. 6A). The release rateof dye from liposomes incubated in the presence of cysteine atconcentrations of 15 μM (solid squares), 75 μM (open triangles), 150 μM(X symbols), 300 μM (open circles), 1500 μM (solid circles), 3000 μM (+symbols), and 15000 μM (open diamonds) is shown.

FIGS. 9A-9C show that the rate of mPEG-MeDTB-lipid cleavage iscysteine-concentration dependent, with a slow rate of cleavage, asevidenced by release of entrapped fluorophore, at cysteineconcentrations of 15-75 μM. In comparing the data in FIG. 9A with thatin FIG. 9B, it is seen that the mPEG-MeDTB-DSPE compound (FIG. 9B)cleaves approximately 10 times more slowly than the mPEG-DTB-DSPEcompound (FIG. 9A). Thus, the rate of cleavage can be tailored accordingto the R moiety (see FIG. 2) in the DTB linkage.

b). In Vivo Characterization

The blood circulation lifetime of liposomes prepared as described inExample 8 and that include a polymer-DTB-lipid conjugate in accord withthe invention was determined in mice. In¹¹¹ was entrapped in theliposomes and the liposomes were administered by intravenous injection.One group of test animals additionally received an injection ofcysteine, the control group of animals additionally received aninjection of saline. Blood samples were taken at various times andanalyzed for the presence of liposomes, as evidenced by the presence ofIn¹¹¹.

FIG. 10 shows the results where the counts per minute (CPM) of In¹¹¹ isshown as a function of time following injection of the liposomes andsaline (open circles) or 200 mM cysteine (solid squares). As seen, thecleavage of the mPEG-DTB-DSPE occurred upon exposure to thenaturally-occurring physiologic conditions, as evidenced by the cleavagein the group of mice treated with saline after administration of theliposomes. Administration of an exogeneous reducing agent, cysteine, tothe mice was effective to increase the rate of cleavage of themPEG-DTB-lipid compound in the time frame from between about 2 hours toabout 8 hours.

Importantly, cleavage of the polymer-DTB-lipid compound of the inventionresults in regeneration of the original lipid in unmodified form. Thisis desirable since unnatural, modified lipids can have undesirable invivo effects. At the same time, the compound is stable when stored inthe absence of reducing agents.

In other studies, not shown here, the blood circulation lifetime ofliposomes containing the mPEG-DTB-lipid were compared to liposomescontaining a polymer-lipid conjugate where the polymer and lipid arejoined by a cleavable aliphatic disulfide bond. Aliphatic disulfidelinkages are readily cleaved in vivo and the blood circulation lifetimeof liposomes having polymer chains grafted to their surface by analiphatic disulfide typically do not have the extended blood circulationlifetime observed for liposomes having stably linked polymer chains. Thedithiolbenzyl linkage of the invention, and in particular the morehindered DTB linkages, are more stable in vivo and achieve a longerblood circulation lifetime than liposomes with polymer chains attachedvia an aliphatic disulfide linkage.

B. Amine-Containing Polypeptide

In another embodiment, the invention includes a compound as describedwith respect to FIG. 1A, where the amine-containing ligand is apolypeptide. A synthetic reaction scheme showing preparation of apolymer-DTB-polypeptide is shown in FIG. 11A, with mPEG as the exemplarypolymer. In general, a mPEG-DTB-leaving group compound is preparedaccording to one the synthetic routes described above in FIGS. 2, 4A and5. The leaving group can be nitrophenyl carbonate or any one of theothers described above. The mPEG-DTB-nitrophenyl carbonate compound iscoupled to an amine moiety in a polypeptide by a urethane linkage. The Rgroup adjacent the disulfide in the compound can be H, CH₃, C₂H₅ or thelike and is selected according to the desired rate of disulfidecleavage.

FIG. 11B shows the decomposition products upon cysteine-mediatedcleavage of the compound. As seen the native protein with nomodification to the protein amine group is regenerated upon cleavage.

Attachment of polymer chains, such as PEG, to a polypeptide oftendiminishes the enzymatic or other biological activity, e.g., receptorbinding, of the polypeptide. However, polymer modification of apolypeptide increases the blood circulation lifetime of the polypeptide.In the present invention, the polymer-modified polypeptide isadministered to a subject. As the polymer-modified polypeptidecirculates exposure to physiologic reducing conditions, such as bloodcysteine and other in vivo thiols, initiates cleavage of the polymerchains from the polypeptide. As the polymer chains are released from thepolypeptide, the biological activity of the polypeptide is graduallyrestored. In this way, the polypeptide initially has a sufficient bloodcirculation lifetime for biodistribution, and over time regains its fullbiological activity as the polymer chains are cleaved.

In a study performed in support of the invention, lysozyme was used as amodel polypeptide and an mPEG-MeDTB-lysozyme conjugate was prepared by asynthetic route similar to those described above. Lysozyme was incubatedwith mPEG-MeDTB-nitrophenylcarbonate in 0.1 M borate, at pH 9 at a 2:1ratio of nitrophenylcarbonate to amino group of lysozyme. Afterreactions times of 15 minutes and 3 hours, samples were characterized bySDS-PAGE. A comparative compound was prepared by reacting lysozyme underthe same conditions for 60 minutes with a conjugate of mPEG-nitrophenylcarbonate, which will form a stable mPEG-lysozyme conjugate.

FIG. 12 shows a rendering of the SDS-PAGE gel. Lane 1 corresponds to thecompound formed after 15 minutes reaction of lysozyme withmPEG-MeDTB-nitrophenylcarbonate and Lane 2 represents the compoundformed after a 1 hour reaction time of the same compounds. Lane 3represents native lysozyme and Lane 4 corresponds to lysozyme reactedfor 1 hour with mPEG-nitrophenylcarbonate. The molecular weight markersin Lane 5 are as follows, from the top down:

Molecular Weight (kDaltons) Marker 1163 β-galactosidase 97.4phosphorylase b 66.3 bovine serum albumin 55.4 glutamic dehydrogenase36.5 lactate dehydrogenase 31 carbonic anhydrase 21.5 trypsin inhibitor14.4 lysozyme

Comparison of Lane 1 and Lane 2 shows that the longer reaction timeresults in an increase in compound molecular weight, consistent withadditional mPEG chains conjugated to the polypeptide at longerincubation time.

Lanes 6-9 of the SDS-PAGE profile correspond to the samples in Lanes 1-4after treatment with 2% β-mercaptoethanol for 10 minutes at 70° C. ThemPEG-MeDTB-lysozyme conjugate after exposure to a reducing agentdecomposed to regenerate native lysozyme, as evidenced by the band inLanes 6 and 7 at 14.4 kDa. In contrast, the stable mPEG-lysozymecompound was not affected upon incubation with a reducing agent, asevidenced by the agreement in the profile in Lane 9 and Lane 4.

Also evident from the SDS-PAGE profile is that covalent attachment ofmPEG-MeDTB to a protein forms a mixture of conjugates containing variousmPEG-protein ratios. This ratio is dependent on the reaction time andconditions. This is clearly seen in viewing the bands in Lanes 1 and 2,where Lane 1 shows lysozyme derivatized with from about 1-6 PEG chains.In Lane 2, the longer reaction time yielded mPEG-MeDTB-lysozymeconjugates with a higher mPEG-protein ratio. All cleavable conjugateswere readily cleaved to regenerate the native protein, as seen in thebands of Lanes 6 and 7.

It will be appreciated that any of the hydrophilic polymers describedabove are contemplated for use. The molecular weight of the polymer isselected depending on the polypeptide, the number of reactive amines onthe polypeptide and the desired size of the polymer-modified compound.

Polypeptides contemplated for use are unlimited and can benaturally-occurring or recombinantly produced polypeptides. Small, humanrecombinant polypeptides are preferred, and polypeptides in the range of10-30 KDa are preferred. Exemplary polypeptides include cytokines, suchas tumor necrosis factor (TNF), interleukins and interferons,erythropoietin (EPO), granulocyte colony stimulating factor (GCSF),enzymes, and the like. Viral polypeptides are also contemplated, wherethe surface of a virus is modified to include one or more polymer chainlinked via a DTB reversible linkage. Modification of a virus containinga gene for cell transfection would extend the circulation time of thevirus and reduce its immunogenicity, thereby improving delivery of anexogeneous gene.

C. Amine-Containing Drug

In yet another embodiment of the invention, a compound of the formpolymer-DTB-amine-containing drug is contemplated. The compound is ofthe structure described above, and in particular with respect to FIG. 1Awhere the amine-containing ligand in the figure is the amine-containingdrug. Modification of therapeutic drugs with PEG is effective to improvethe blood circulation lifetime of the drug and to reduce anyimmunogenicity.

A polymer-DTB-amine-containing drug is prepared according to any of thereaction schemes described above, with modifications as necessary toprovide for the particular drug. A wide variety of therapeutic drugshave a reactive amine moiety, such as mitomycin C, bleomycin,doxorubicin and ciprofloxacin, and the invention contemplates any ofthese drugs with no limitation. It will be appreciated that theinvention is also useful for drugs containing an alcohol or carboxylmoiety. In the case where the drug contains a hydroxyl or carboxylmoiety suitable for reaction, the polymer-DTB moiety can be linked tothe drug via urethane, ester, ether, thioether or thioester linkages. Inall of these embodiments, the polymer-DTB-drug compound afteradministration in vivo thiolytically decomposes to regenerate theamine-containing drug in its native, active form, therapeutic activityof the compound after modification and prior to administration is notnecessary. Thus, in cases where modification of the drug with theDTB-polymer causes a reduction or loss of therapeutic activity, afteradministration and cleavage of the DTB-polymer from the drug, activityof the drug is regained.

In studies performed in support of the invention, the drug nitroanilidewas reacted with mPEG-MeDTB-nitrophenylcarbonate to form anmPEG-MeDTB-para-nitroanilide compound, as shown in FIG. 13.Decomposition of the compound upon exposure to a reducing agent yieldsthe products shown in the figure, with the drug para-nitroanilideregenerated in an unmodified state.

The mPEG-MeDTB-para-nitroanilide compound was incubated in vitro inbuffer containing 5 mM cysteine and the absorbence of samples withdrawnat various times is shown in FIG. 14A. Seen in the figure are samplesmeasured at the following time points: time zero (closed diamonds), 2minutes (closed squares), 5 minutes (x symbols), 10 minutes (opensquares), 20 minutes (triangles), 40 minutes (open diamonds) and 80minutes (closed circles). The change in the UV spectra as a function ofincubation time in cysteine is evident, showing cysteine-mediatedrelease of para-nitroanilide from the mPEG-MeDTB-para-nitroanilidecompound.

FIG. 14B shows the amount of para-nitroanilide, in mole/L, released invitro from the mPEG-MeDTB-para-nitroanilide conjugate incubated in thepresence of 5 mM cysteine (closed circles), 1 mM cysteine (closedsquares) and 0.15 mM cysteine (closed diamonds). The rate of drugrelease from the conjugate was dependent on the concentration ofreducing agent present.

From the foregoing, it can be seen how various objects and features ofthe invention are met. The compounds of the invention comprise anamine-containing ligand reversibly joined to a hydrophilic polymer viaan ortho or para-disulfide of a benzyl urethane linkage. This linkagewhen subjected to mild thiolytic conditions is cleaved to regenerate theoriginal amine-containing ligand in its unmodified form. The rate ofcleavage can be controlled by steric hinderance of the disulfide in thelinkage and/or by controlling the thiolytic conditions in vivo. Thecompounds prior to cleavage of the dithiobenzyl linkage are providedwith an increased blood circulation lifetime, improved stability andreduced immunogenicity.

III. Examples

The following examples further illustrate the invention described hereinand are in no way intended to limit the scope of the invention.

Materials

All materials were obtained from commercially suitable vendors, such asAldrich Corporation.

Example 1 Synthesis of mPEG-DTB-DSPE

mPEG-MeDTB-nitrophenylcarbonate (300 mg, 0.12 mmol, 1.29 eq) wasdissolved in CHC₃ (3 ml). DSPE (10 mg, 0.093 mol) and TEA (58.5 μl, 0.42mmol, 4.5 eq) were added to PEG-solution, and was stirred at 50° C. (oilbath temp). After 15 minutes, TLC showed that the reaction didn't go tocompletion. Then two portions of TEA (10 μl, and 20 μl), and fewportions of mPEG-MeDTB-nitrophenylcarbonate (50 mg, 30 mg, 10 mg) wereadded every after 10 minutes, until the reaction went to completion.Solvent was evaporated. Product mixture was dissolved in MeOH, and 1 gof C8 silica was added. Solvent was evaporated again. Product containingC8 silica was added on the top of the column, and was eluted withMeOH:H₂O gradient (pressure), MeOH:H₂O=30:70, 60 ml; MeOH:H₂O=50:50, 60ml; MeOH:H₂O=70:30, 140 ml (starting material eluted); MeOH:H₂O=75:25=40ml; MeOH:H₂O=80:20, 80 ml (product eluted); MeOH:H₂O=85:15, 40 ml;MeOH:H₂O=90:10, 40 ml: MeOH=40 ml; CHCl₃:MeOH:H₂O=90:18:10, 40 ml.Fractions containing pure product were combined and evaporated to giveproduct as colorless thick liquid. Tertiary butanol (5 ml) was added toit, lyophilized and the dried in vacuo over P₂O₅ to give product aswhite fluffy solid (252 mg, 89% yield).

The ortho- and para-DTB-DSPE compounds were purified by silica gelchromatography (methanol gradient 0-10% in chloroform, ≈70% isolatedyield) and the structures confirmed by NMR and MALDI-TOFMS. (¹H NMR forpara conjugate: (d6-DMSO, 360 MHz) δ 0.86 (t, CH₃, 6H), 1.22 (s, CH₂ oflipid, 56H), 1.57 (m, CH₂CH₂CO₂, 4H), 2.50 (2×t, CH₂CO₂, 4H), 2.82 (t,CH₂S, 2H), 3.32 (s, OCH₃, 3H), 3.51 (m, PEG, ≈180H), 4.07 (t,PEG-CH₂OCONH, 2H), 4.11 & 4.28 (2×dd CH₂CH of glycerol, 2H), 4.98 (s,benzyl-CH₂, 2H), 5.09 (m, CHCH₂ of lipid), 7.35 & 7.53 (2×d, aromatic,4H) ppm. The ortho conjugate differed only in benzyl and aromaticsignals at 5.11 (s, CH₂, 2H), and 7.31 (d, 1H), 7.39 (m, 2H) 7.75 (d,1H) ppm.

MALDI-TOFMS produced a distribution of ions spaced at equal 44 Daintervals, corresponding to the ethylene oxide repeating units. Theaverage molecular weights of the compounds was 3127 and 3139 Da for paraand ortho isomers respectively (theoretical molecular weight≈3100 Da).

The reaction scheme is illustrated in FIG. 2.

Example 2 Synthesis of mPEG-DTB-DSPE

A. mPEG-MeDTB-DSPE

This reaction scheme is illustrated in FIGS. 4A-4B.

mPEG(5K)-OH (40 g, 8 mmol) was dried azeotropically with toluene (totalvolume was 270 ml, 250 ml was distilled off by Dean-Stark).Dichloromethane (100 ml) was added to mPEG-OH. P-nitrophenylchloroformate (2.42 g, 12 mmol, 1.5 eq), and TEA (3.3 ml, 24 mmol, 3 eq)were added to PEG solution at 4° C. (ice water), while takingprecautions against moisture. Light yellow TEA hydrochloride salt wasformed. After 15 minutes cooling bath was removed, and the reactionmixture was stirred at room temperature overnight. TLO showed(CHCl₃:MeOH:H₂O=90:18:2) that the reaction was complete. Solvent wasevaporated. The residue was dissolved in ethyl acetate (˜50° C.). TEAhydrochloride salt was filtered off and washed with warm ethyl acetate.Solvent was evaporated and the product recrystallized with isopropanol(three times). Yield: 38.2 g (92%). ¹H NMR (DMSO-d₆, 360 MHz) δ 3.55 (s,PEG, 450H); 4.37 (t, PEG-CH₂, 2H); 7.55 (d, C₆H₅, 2H); 8.31 (d, C₆H₅,2H).

1-Amino-2-propanol (1.1 ml, 14.52 mmol, 3 eq), and TEA (2.02 ml, 14.52mmol, 3 eq) were added to mPEG(5K)-nitrophenyl carbonate (25 g, 4.84mmol) in DMF (60 ml) and CH₂Cl₂ (40 ml). It was a yellow clear solution.The reaction mixture was stirred at room temperature for 30 minutes. TLC(CHC1₃:MeOH=90:10) showed that the reaction went to completion. Solvent(dichloromethane) was evaporated. Isopropanol (250 ml) was added to theproduct mixture in DMF (60 ml). Product precipitated immediately, andthen recrystallized with iPrOH (three times). Yield: 22.12 g (90%). ¹HNMR (DMSO-d₆, 360 MHz) δ 0.98 (d, CH₃CH(OH)CH₂, 3H); 3.50 (s, PEG,180H); 4.03 (t, PEG-CH₂, 2H); 4.50 (d, CH₃CHOH, 1H); 7.0 (t,mPEG-OCONH).

mPEG(5K)-urethane-2-methyl propanol (22.12 g, 4.34 mmol) was driedazeotropically with toluene (45 ml). Dichloromethane (60 ml) was addedto it. Methane sulfonyl chloride (604.6 μl, 7.81 mmol, 1.8 eq) and TEA(3.93 ml, 28.21 mmol, 6.5 eq) were added to mPEG-solution at 0° C. whilemaintaining stirring and taking precautions against moisture. After 30minutes, cooling bath was removed, and the reaction mixture was stirredat room temperature for 16 h. Solvent was evaporated. Ethyl acetate wasadded to remove TEA salts. The product was recrystallized withisopropanol (three times). Yield: 20.27 g (90%). ¹H NMR (DMSO-d₆, 360MHz) δ 1.27 (d, CH₃CHOSO₂CH₃, 3H); 3.162 (s, CH₃O₂SOCH, 3H); 3.50 (s,PEG, 180H); 4.07 (t, PEG-CH₂, 2H); 4.64 (q, CH₃CHOH, 1H); 7.43 (t,mPEG-OCONH).

mPEG(5K)-urethane-2-methyl-methane sulfone (10.27 g, 1.98 mmol) wasdried azeotropically with toluene (20 ml, each time). Sodium hydride(377 mg, 9.4 mmol, 4.75 eq) was added in anhydrous toluene (60 ml) at 0°C. (in ice water). After 5 minutes, triphenylmethanethiol (3.92 g, 14.6mmol, 7.15 eq) was added to the solution. After 10 minutes,mPEG-urethane-2-methyl-methane sulfone (10.27 gm, 1.98 mmol) was addedto the reaction mixture. It became a yellow solution. After 45 minutes,TLC (CHCl₃:MeOH:H₂O=90:18:2) showed that the reaction went tocompletion. Acetic acid (445.57 μl, 7.42 mmol, 3.75 eq) was added to thereaction mixture to neutralize excess of sodium hydride. The solutionbecame thick and whitish. Solvent was evaporated and the solid wasrecrystallized with ethyl acetate (30 ml) and isopropanol (70 ml). Theproduct mixture did not dissolve completely, while precipitate filteredoff. Then the product mixture was recrystallized withisopropanol/tert-butyl alcohol (100 ml/20 ml). Yield: 8.87 g (84%). ¹HNMR (DM50-d₆, 360 MHz) δ 0.74 (d, CH₃CHSC(C₆H₅)₃, 3H), 3.50 (s, PEG,180H), 4.0 (t, PEG-CH₂, 2H), 4.64 (q, CH₃CHOH, 1H); 7.49 (t,mPEG-OCONH); 7.20-7.41 (m, SC(C₆H₅)₃, 15H).

mPEG(5K)-urethane-2-methyl-triphenylmethanethiol (8.87 g, 1.65 mmol) wasdissolved in TFA/CH₂C1₂ (10 ml/10 ml) at 0° C. Under vigorous stirring,methoxy carbonylsulfenyl chloride (185.5 μl, 1.99 mmol, 1.2 eq) wasadded to the solution. The reaction mixture was stirred at roomtemperature for 15 minutes. TLC (CHCl₃:MeOH=90:10) showed that thereaction was complete. Solvents were evaporated. The product mixture wasrecrystallized with isopropanol:tert-butyl alcohol (80 ml:20 ml) twotimes. Tertiary butanol (5 ml) was added to the product, which was thenlyophilized and dried in vacuo over P₂O₅ to give product as white fluffysolid (8.32 g, 97% yield). ¹H NMR (DMSO-d₆, 360 MHz) δ 1.17 (d,CH₃CHSSCOOCH₃, 3H); 3.42 (s, PEG, 180H); 3.84 (s, CH₃OCOSSCH, 3H); 4.05(t, mPEG-CH₂, 2H); 7.38 (t, mPEG-OCONH, 1H).

mPEG(5K)-urethane ethyl(methyl)dithiocarbonyl methoxide (8.32 g, 1.6mmol) was dissolved in dry methanol (20 ml), and chloroform (2.5 ml). Asolution of mercapto benzyl alcohol (592 mg, 4 mmol, 2.5 eq) in drymethanol (2 ml) was added to the PEG-solution. The reaction mixture wasstirred at room temperature for 18 h. Solvent was evaporated, productmixture was recrystallized with ethyl acetate/isopropanol, 30 ml/100 ml(3 times). NMR showed ˜16% product was formed. So, another portion ofmercapto benzyl alcohol (322 mg, 2.18 mmol, 1.8 eq) in MeOH (2 ml) wasadded dropwise to the product mixture in MeOH/CHCl₃ (24 ml/l ml) at 0°C. (ice water). After addition (˜10 minutes) completion, ice bath wasremoved, and the reaction mixture was stirred at room temperature for 24h. TLC (CHCl₃:MeOH:H₂O=90:18:2) showed that the reaction was complete.Solvent was evaporated, and then product mixture was recrystallized withethyl acetate/isopropanol, 30 ml/100 ml. Yield: 7.25 g, (94%). ¹H NMR(DMSO-d₆, 360 MHz) δ 1.56 (d, CH₃CHSSC₆H₅CH₂OH, 3H); 3.29 (CH₃O-PEG,3H); 3.50 (s, PEG, 450H); 4.03 (t, mPEG-CH₂, 2H); 4.46 (d, HOCH₂C₆H₅,2H); 5.16 (t, HOCH₂C₆H₅, 1H); 7.30 (d, C₆H₅, 2H); 7.40 (br t,mPEG-OCONH, 1H); 7.50 (d, C₆H₅, 2H).

mPEG(5K)-urethane-ethyl(methyl)-dithiobenzyl alcohol (6.75 g, 1.27 mmol)was dissolved in CHCl₃ (30 ml), P-nitrophenyl chloroformate (513 mg,2.54 mmol, 2 eq) was added to it at 0° C. (ice water). After 5 minutestriethylamine (531 μl, 3.81 mmol, 3 eq) was added. After 30 minutes icebath was removed, and the reaction mixture was stirred at roomtemperature overnight. Solvent was evaporated. The product mixture wasdissolved in ethyl acetate. TEA salt was filtered off, and then solventwas evaporated. Then the product mixture was recrystallized with ethylacetate/isopropanol, 30 ml/100 ml (three times). Yield: 6.55 g (94%). ¹HNMR (DMSO-d₆, 360 MHz) δ 1.17 (d, CH₃CHSSC₆H₅, 3H); 3.24 (CH₃O-PEG, 3H);3.40 (s, PEG, 180H); 4.03 (br t, mPEG-CH₂, 2H); 5.28 (S, C₆H₅CH₂OCO,2H); 7.45-8.35 (m, C₆H₅)₂, 8H)

mPEG-MeDTB-nitrophenylcarbonate (766 mg, 0.14 mmol, 1.29 eq) wasdissolved in CHC1₃ (5 ml). DSPE (70 mg, 0.093 mol) and TEA (58.5 pd,0.42 mmol, 4.5 eq) were added to PEG-solution, and was stirred at 50° C.(oil bath temp). After 20 minutes, TLC showed that the reaction didn'tgo to completion. More mPEG-MeDTB-nitrophenylcarbonate (total 1239 mg,0.23 mmol, 2.47 eq) and 1-hydroxybenztriazole (HOBt) (25 mg, 0.19 mmol,2 eq) were added. After 20 minutes, TLC(CHC1₃:MeOH: H₂O=90:18:2, withmolybdenum and ninhydrin) showed that the reaction was complete. Solventwas evaporated. Product mixture was dissolved in warm (42° C.) ethylacetate. It was a cloudy solution (TEA salt precipitated). The solutionwas filtered, and solvent was evaporated. MeOH and 2 g of C8 silica wasadded to the product mixture. Solvent was evaporated again. Productcontaining C8 silica was added on the top of the column, and was elutedwith MeOH:H₂O gradient (pressure), MeOH:H₂O=30:70, 100 ml;MeOH:H₂O=50:50, 100 ml; MeOH:H₂O=70:30, 250 ml (starting materialeluted); MeOH:H₂O=75:25, 40 ml; MeOH:H₂O=80:20, 200 ml (product eluted);MeOH=100 ml; CHCl₃:MeOH:H₂O=90:18:2, 100 ml; CHCl₃:MeOH:H₂O=75:36:6, 100ml. Fractions containing pure product were combined and evaporated togive product as colorless thick liquid. Tertiary butanol (5 ml) wasadded to it, lyophilized and then dried in vacuo over P₂O₅ to giveproduct as white fluffy solid (467 mg, 83% yield). ¹NMR (DMSO-d₆, 360MHz) δ 0.83 (d, 2 (CH₃), 3H); 1.16 (d, CH₃CHSSC6H₅, 3H); 1.21 (s, 28(CH₂, 56H); 1.47 (br m, CH₂CH₂CO, 4H); 2.23 (2×t, CH₂CH₂CO, 4H); 3.50(s, PEG, 180H); 4.04 (br t, mPEG-CH₂, 2H); 4.05 (trans d, PO₄CH₂CHCH₂,1H); 4.24 (cis d, PO₄CH₂CHCH₂, 1H); 4.97 (s, C₆H₅CH₂OCO-DSPE, 2H); 5.03(br s, (PO₄CH₂CH, 1H); 7.32 (d, C₆H₅, 2H); 7.53 (d, C₆H₅, 2H); 7.52 (brs, mPEG-OCONH, 1H). MALDI-TOFMS produced a bell shaped distribution ofions spaced at equal 44 Da intervals, corresponding to the ethyleneoxide repeating units. The average molecular mass of the conjugate andmPEG-thiol (mostly cleaved disulfide) is 6376 and 5368 Da (theoreticalmolecular mass ˜6053, and 5305 Daltons).

B. mPEG-ethylDTB-DSPE

mPEG-urethane ethyl(ethyl)dithiocarbonyl methoxide (2 g, 0.90 mmol) wasdissolved in dry methanol (8 ml). At the beginning the solution wascloudy, but after 5 minutes it became a clear solution. Mercaptobenzylalcohol (265.2 mg, 1.79 mmol, 2 eq) was added to the PEG-solution. Thereaction mixture was stirred at room temperature for 30 hours. Ether (70ml) was added to the reaction solution to precipitate the product, andkept at 4° C. overnight. The white solid was filtered and recrystallizedwith ethyl acetate/ether, 30 ml/70 ml. Yield: 1.96 g, (94%). ¹H NMR(DMSO-d₆, 360 MHz) δ0.86 (d, CH₃CH₂CHSSC₆H₅CH₂OH, 3H); 1.42 (p,CH₃CH₂CHSSC₆H₅CH₂OH, 1H); 1.64 (p, CH₃CH₂CHSSC₆H₅CH₂OH, 1H); 3.51 (s,PEG, 180H); 4.03 (t, mPEG-CH₂, 2H); 4.47 (d, HOCH₂C₆H₅, 2H); 5.20 (t,HOCH₂C₆H₅, 1H); 7.31 (d, C₆H₅, 2H); 7.42 (br t, mPEG-OCONH, 1H); 7.49(d, C₆H₅, 2H).

N-hydroxy-s-norbornene-2,3-dicarboxylic acid imide (HONB) (48 mg, 0.269mmol) was added to DSPE (55 mg, 0.073 mmol) in CHCl₃ (3 ml) at 50° C.(oil bath temperature). After 3-4 minutes it became a clear solution.Then mPEG-EtDTB-nitrophenylchloroformate (334 mg, 0.134 mmol) was added,followed by triethylamine (TEA, 45 μl, 0.329 mmol). After 20 minutes TLC(CHCl₃:MeOH:H₂O=90:18:2) showed that the reaction went to completion(molybdenum and ninhydrin sprays). Solvent was evaporated. Productmixture was dissolved in methanol, mixed with C8 silica (1 g) andstriped of the solvent by rotary evaporation. The solid residue wasadded on the top of the C8-column, which was then eluted with MeOH:H₂Ogradient (pressure), MeOH:H₂O=30:70, 60 ml; MeOH:H₂O=50:50, 60 ml;MeOH:H₂O=70:30, 140 ml; MeOH:H₂O=75:25=140 ml (starting materialeluted); MeOH:H₂O=80:20, 80 ml; MeOH:H₂O=90:10, 140 ml (product eluted);MeOH=40 ml; CHCl₃:MeOH:H₂O=90:18:10, 40 ml. Fractions containing pureproduct were combined and evaporated to give product as colorless thickliquid. Tertiary butanol (5 ml) was added, lyophilized and then dried invacuo over P₂O₅ to give product as white fluffy solid (175 mg, 78%yield). ¹H NMR (DMSO-d₆, 360 MHz) δ 0.85 (d, 2 (CH₃), 6H; d,CH₃CHSSC₆H₅, 3H); 1.22 (s, 28 (CH₂), 56H); 1.49 (br m, CH₂CH₂CO, 4H);2.24 (2×t, CH₂CH₂CO, 4H); 3.50 (s, PEG, 180H); 4.04 (br t, mPEG-CH₂,2H); 4.08 (trans d, PO₄CH₂CHCH₂, 1H); 4.27 (cis d, PO₄CH₂CHCH₂, 1H);4.98 (s, C₆H₅CH₂OCO-DSPE, 2H); 5.06 (br s, (PO₄CH₂CH, 1H); 7.34 (d,C₆H₅, 2H); 7.53 (d, C₆H₅, 2H); 7.55 (br s, mPEG-OCONH, 1H).

Example 3 Synthesis of mPEG-DTB-nitrophenylchloroformate

A. Procedures for Synthesis of 1-(mercaptomethyl)ethylammonium chloride

1. 2-Amino-1-methylethyl hydrogen sulfate. 1-Amino-2-propanol (22.53 g,0.3 mol) was vigorously stirred in an ice bath. Sulfuric acid (16.10 ml,0.3 mol) was added very slowly, over the course of one hour. Thickvapors and a very viscous solution were formed in the flask. Afteraddition was complete, the reaction was heated between 170° C. and 180°C., under reduced pressure, connected to the house vacuum. Upon heating,the reaction turned light brown. After all water was removed(approximately 1 hour) it was allowed to cool to room temperature. Uponcooling a brown, glassy solid was formed which would crystallize whentriturated with methanol. It was dissolved in water (50 ml) at 60° C.Enough warm methanol was added to make the solution 80% methanol. Uponcooling, crystals formed which were then filtered and dried over P₂O₅.Yield: 17.17 g (37%). ¹H NMR (D₆-DMSO): δ 1.16 (d, CH₃, 3H); δ 2.78 (dd,NH₃—CH₂, 1H); δ 2.97 (dd, NH₃—CH₂, 1H); δ 4.41 (m, CH—OSO₃, 1H); δ 7.69(s, H₃N, 3H). Melting point: 248°-250° C. (lit: 250° C.)

2. 5-Methylthiazolidine-2-thione. 2-Amino-1-methylethyl hydrogen sulfate(23.03 g, 148 mmol) and carbon disulfide (10.71 ml, 178 mmol, 1.2 eq.)were stirred in a 250 ml round-bottom-flask in 50% aqueous ethanol (40ml). To this, sodium hydroxide (13.06 g, 327 mmol, 2.2 eq.) in 50%aqueous ethanol (50 ml) was added drop-wise, very slowly. Upon additionof sodium hydroxide, all starting materials dissolved and the solutionturned orange. The reaction was refluxed (85° C.) for 40 minutes, afterwhich time it turned bright yellow and a thick precipitate was formed.Ethanol was evaporated and then the aqueous solution was warmed and thenfiltered through a Buchner funnel to remove all water-solubleimpurities. The remaining crystals were dissolved in warm ethanol andthen warm water was added until the solution was 80% water. The mixturewas allowed to cool and then refrigerated, yielding long, needle-likecrystals. Yield: 14.64 g (75%). ¹H NMR (D₆-DMSO): δ 1.33 (d, CH₃, 3H); δ3.50 (m, R₃CH, 1H); δ 3.95 (dd, N—CH₂, 1H); δ 4.05 (m, N—CH₂, 1H); δ10.05 (s, NH, 1H). Melting point: 92.5-93.5 (lit: 94-95).

3. 1-(mercaptomethyl)ethylammonium chloride.5-Methylthiazolidine-2-thione (6.5 g, 49 mmol) was placed in a 250 mlround-bottom-flask. A solution of aqueous hydrochloric acid (40 ml, 18%in H₂O) was added and the flask was heated in an oil bath. The reactionrefluxed (120° C.) for one week. Three times throughout the week 1 ml ofconcentrated hydrochloric acid was added. The reaction was monitoredusing TLC with ethyl acetate as eluent. They were visualized using UV,ninhydrin, and iodine vapors. Through most of the week the reaction wasa heterogeneous mixture, with the starting material as oil which wasdenser than water. After one week the oil starting material was gone,although still visible on TLC. The reaction was removed from heat andallowed to cool to room temperature, and then was refrigerated tocrystallize starting material. The crystallized starting material wasfiltered. Filtrate was evaporated and it was dried over P₂O₅ and NaOH toremove all water and HCl. The crude product was washed with two portionsof diethyl ether (50 ml each) to remove all starting material. It wasagain dried over P₂O₅. Yield: 2.83 g (45%). ¹H NMR (D₆-DMSO): δ 1.33 (d,CH₃, 3H); δ 2.92 (m, N—CH₂, 2H); δ 3.12 (m, SH, 1H); δ 3.18 (m, R₃—CH,1H); δ 8.23 (bs, NH₃, 3H). Melting point: 80-82° C. (lit: 92-94).

The reaction scheme is illustrated in FIG. 5.

B. Synthesis of mPEG-ethyl-DTB-nitrophenylchloroformate

1. 2-Amino-1-ethylethyl hydrogen sulfate. 1-Amino-2-butanol (15 ml, 158mmol) was vigorously stirred in a 100 ml round-bottom-flask in an icebath. Sulfuric acid (8.43 ml, 158 mmol) was added very slowly, over thecourse of one hour. Thick vapors and a very viscous solution were formedin the flask. After addition was complete, the reaction was heatedbetween 170° and 180° C., under reduced pressure, connected to the housevacuum. Upon heating, the reaction turned light brown. After all waterwas removed (approximately 1 hour) it was allowed to cool to roomtemperature. Upon cooling a brown, glassy solid was formed. It wasdissolved in hot water (50 ml) and then placed in the refrigeratorovernight. Upon cooling, crystals formed which were then filtered anddried over P₂O₅. Yield: 9.98 g (37%). ¹H NMR (D₆-DMSO): δ 0.87 (t, CH₃,3H); δ 1.51 (q, CH₃—CH₂, 2H); δ 2.82 (dd, NH₃—CH₂, 1H); δ 3.00 (dd,NH₃—CH₂, 1H); δ 4.21 (m, CH—OSO₃, 1H); δ 7.70 (s, H₃N, 3H).

2. 5-Ethylthiazolidine-2-thione. 2-Amino-1-ethyl-ethyl hydrogen sulfate(9.98 g, 59 mmol) and carbon disulfide (4.26 ml, 71 mmol, 1.2 eq.) werestirred in a 100 ml round-bottom-flask in 50% aqueous ethanol (15 ml).To this, sodium hydroxide (5.20 g, 130 mmol, 2.2 eq.) in 50% aqueousethanol (20 ml) was added drop-wise, very slowly. Upon addition ofsodium hydroxide, all starting materials dissolved and the solutionturned orange. The reaction was refluxed (85° C.) for 40 minutes, afterwhich time it turned bright yellow and a thick precipitate was formed.Ethanol was evaporated and then the aqueous solution was warmed and thenfiltered through a Buchner funnel to remove all water-solubleimpurities. The remaining crystals were dissolved in warm ethanol andthen warm water was added until the solution was 80% water. The mixturewas allowed to cool and then refrigerated, yielding needle-likecrystals. Yield: 7.28 g (86%). ¹H NMR (D₆-DMSO): δ 0.88 (t, CH₃, 3H); δ1.66 (in, CH₃—CH₂, 2H); δ 3.58 (m, R₃CH, 1H); δ 3.93 (m, N—CH₂, 2H); δ10.06 (s, NH, 1H). Melting point: 76-78° (lit: 76.6-76.9).

3. 1-(mercaptoethyl)ethylammonium chloride. 5-Ethylthiazolidine-2-thione(7.24 g, 50 mmol) was placed in a 250 ml round-bottom-flask. A solutionof aqueous hydrochloric acid (45 ml, 18% in H₂O) was added and the flaskwas heated in an oil bath. Upon heating, the starting material melted,forming, all heterogeneous mixture. The reaction refluxed (120° C.) forone week. Four times throughout the week 1 ml of concentratedhydrochloric acid was added. The reaction was monitored using TLC withethyl acetate as eluent. They were visualized using UV, ninhydrin, andiodine vapors. Throughout the week the reaction was a heterogeneousmixture, with the starting material as oil which was denser than water.The reaction was removed from heat and allowed to cool to roomtemperature, and then was refrigerated to crystallize starting material.The crystallized starting material was filtered. Filtrate was evaporatedand it was dried over P₂O₅ and NaOH to remove all water and HCl. Thecrude product was washed with two portions of diethyl ether (50 ml each)to remove all starting material. It was again dried over P₂O₅. Yield:3.60 g (52%).

The reaction scheme is illustrated in FIG. 5.

Example 4 Synthesis of mPEG-DTB-lipid

1,2-distereoyl-sn-glycerol (500 mg, 0.8 mmol) was dried azeotropicallywith benzene (3 times). Para-nitrophenyl chloroformate (242 mg, 1.2mmol, 1.5 eq), dimethylaminopyridine (DMAP) (10 mg, 0.08 mmol, 0.1 eq),and TEA (334.5 μl, 2.4 mmol, 3 eq) were added to 1,2-distereoyl glycerolin CHCl₃ (5 ml). The reaction mixture was stirred at room temperaturefor 2 h. TLC (Toluene:ethyl acetate=7:3) showed that the reaction wascomplete. Then the product mixture was extracted with 10% citric acid toremove dimethylaminopyridine (DMAP), washed with acetonitrile (3 ml, 4times) to remove excess of p-nitrophenyl chloroformate. Pure product wasdried in vacuo over P₂O₅. Yield: 557 mg(88%). ¹H NMR (CHCl₃, 360 MHz) δ0.88 (t, end CH₃, 6H); 1.25 (s, 28×CH₂, 56H); 1.58 (m, CH₂CH₂CO, 4H);2.34 (2×t, CH₂CO, 4H); 4.22 (trans d, CH₂OCOC₁₇H₃₅, 1H); 4.35 (m,OCOOCH₂CH, 2H); 4.51 (cis d, CH₂OCOC₁₇H₃₅, 1H); 5.37 (m, OCOOCH₂CH, 1H);7.39 (d, C₆H₅, 2H); 8.28 (d, C₆H₅, 2H).

Ethylene diamine (42 μl, 0.63 mmol, 5-fold excess), and pyridine (200μl), were added in CHCl₃ (1 ml). 2-disteroyl-sn-p-nitrophenyl carbonate(100 mg, 0.13 mmol) was dissolved in CHCl₃(1 ml) and added dropwise toethylene diamine solution with a pastuer pipette at 0° C. (ice water)and continued overnight (16 h). TLC (CHCl₃:MeOH:H₂O=90:18:2 andCHCl₃:MeOH 90:10) showed that the reaction was complete. Solvent wasevaporated to remove pyridine. Then the product mixture was dissolved inCHCl₃, loaded onto the column (Aldrich, Silica gel, 60° A, 200-400mesh), and eluted with CHC₃:CH₃COCH₃ and CHCl₃:MeOH gradient,CHCl₃CH₃COCH₃=90:10, 60 ml (upper spot eluted); CHCl₃:MeOH=90:10, 60 ml(product eluted). Fractions containing pure product were combined andevaporated. Tert-butanol was added and dried in vacuo over P₂O₅. Yield:64 mg (75%). ¹H NMR (DMSO-d₆, 360 MHz) δ 0.83 (t, end CH₃, 6H); 1.22 (a,28×CH₂, 56H); 1.51 (m, CH₂CH₂CO, 4H); 2.25 (2×t, CH₂CO, 4H); 2.83 (m,H₂NCH₂CH₂NH, 2H); 3.21 (m, H₂NCH₂CH₂NH, 2H); 4.10-4.14 (m & cis d,COOCH₂CHCH₂, 4H); 5.17 (m, OCOOCH₂CH, 1H); 7.78 (m, H₂NCH₂CH₂NH, 2H).

mPEG-MeDTB-nitrophenylchloroformate (400 mg, 0.162 mmol, 2.2 eq) wasdissolved in CHCl₃ in (2 ml). 1,2-steroyl-sn-ethylene amine (51 mg,0.075 mmol) and TEA (37 μl, 0.264 mmol, 3.52 eq) were added to thesolution. Then the reaction mixture was stirred at 45° C. for 20minutes. TLC (CHCl₃:MeOH:H₂O=90:18:2 and CHCl₃:MeOH=90:10 showed thatthe reaction went to completion. Solvent was evaporated. The productmixture was dissolved in methanol. 2 g of C8 silica was added and thensolvent was evaporated. C8 silica containing product mixture was addedon the top of the C8 column ((Supelco, Supel clean. Lot no. SP0824), andwas eluted with MeOH:H₂O gradient (pressure), MeOH:H₂O=60:40, 40 ml;MeOH:H₂O=70:30, 80 ml (starting material eluted); MeOH:H₂O=80:20, 40 ml;MeOH:H₂O=90:10, 20 ml; CHCl₃:MeOH=5:80:15, 20ml:CHCl₃:MeOH:H₂O=90:18:10, 40 ml (product eluted). Fractions containingpure product were combined and evaporated to give product as colorlessthick liquid. Tertiary butanol (5 ml) was added and the solution waslyophilized and then dried in vacuo over P₂O₅ to give product as whitesolid (200 mg, 89% yield). ¹H NMR (DMSO-d₆, 360 MHz) δδ 0.83 (t, endCH₃, 6H); 1.22 (s, 28×CH₂, 56H); 1.48 (m, CH₂CH₂CO, 4H); 2.25 (2×t,CH₂CO, 4H); 3.10 (m, HNCH₂CH₂NH, 4H); 3.50 (s, PEG, 180H); 4.04 (t,mPEG-CH₂, 2H); 4.09 (trans d, OOOCH₂CHCH₂, 1H); 4.25 (cis d,COOCH₂CHCH₂, 1H); 4.98 (s, C₆H₅CH₂OCO, 2H); 5.23 (m, COOGH₂CHCH₂, 1H);7.18 (m, NHCH₂CH₂NH, 2H); 7.33 (d, C₆H₅, 2H); 7.38 (m, mPEG-OCONH, 1H);7.52 (d, C₆H₅, 2H).

The reaction scheme is illustrated in FIG. 6A.

Example 5 In Vitro Cleavage of mPEG-DTB-DSPE Compound

Ortho-mPEG-DTB-DSPE and para-mPEG-DTB-DSPE (prepared as described inExample 1) were added to a buffered aqueous solution (pH 7.2) in thepresence and absence of 150 μM cysteine. Disappearance of the conjugateswas monitored by HPLC (Phenomenex C₈ Prodigy, 4.6×50 mm column,detection at 277 nm, mobile phase methanol/water 95:5 with 0.1%trifluoroacetic acid at 1 mL/min). The results are illustrated in FIG.7A where the ortho-conjugate is represented by the open circles and thepara-conjugate by the open squares.

Example 6 In Vitro Cleavage of o- and p-mPEG-DTB-DSPE Compound inLiposomes

A. Liposome Preparation

The lipids partially hydrogenated phosphatidylcholine (PHPC),cholesterol and ortho- or para-mPEG-DTB-DSPE (prepared as described inExample 1, mPEG MW=1980 Daltons) were dissolved in a 95:5:3 molar ratio,respectively, in a suitable organic solvent, typicallychloroform/methanol in a 1:1 or 1:3 ratio. The solvent was removed byrotary evaporation to form a dried lipid film. The film was hydratedwith aqueous buffer to from liposomes that were sized via extrusion toan average diameter of 120 nm.

B. In vitro Characterization

The liposomes were incubated in phosphate buffered saline, pH 7.2,containing 5 mM EDTA at 37° C. in the presence of 150 μM cysteine.Disappearance of the conjugates was monitored by HPLC (Phenomenex C₈Prodigy, 4.6×50 mm column, detection at 277 nm, mobile phasemethanol/water 95:5 with 0.1% trifluoroacetic acid at 1 mL/min). Resultsare shown in FIG. 7B where the liposomes comprising the ortho-conjugateare represented by the solid circles and liposomes comprising thepara-conjugate by the solid squares. The open circles and the opensquares correspond to ortho-mPEG-DTB-DSPE and para-mPEG-DTB-DSPE inmicellar form (discussed above in Example 5, FIG. 7A).

Example 7 In Vitro Cleavage of o- and p-mPEG-DTB-DSPE Compound inLiposomes

A. Liposome Preparation

The lipids dioleoyl phosphatidylethanolamine (DOPE) and ortho- orpara-mPEG-DTB-DSPE (prepared as described in Example 1, mPEG MW=1980Daltons) were dissolved a 97:3 molar ratio in chloroform/methanol 1:1.The solvent was removed by rotary evaporation to form a dried lipidfilm. The lipid film was hydrated with an aqueous solution containing 30mM each of the fluorophores p-xylene-bis-pyridinium bromide andtrisodium 8-hydroxypyrenetrisulfonate was hydrated with aqueous bufferto form liposomes that were sized via extrusion to an average diameterof 100 nm.

B. In Vitro Characterization

The liposomes were incubated in HEPES buffer, pH 7.2, at 37° C. in thepresence of cysteine at concentrations of 15 μM, 150 μM, 300 μM and 1.5mM. Percent of released dye was determined as the increase in samplefluorescence (λ_(em)=512 nm, λ_(ex)=413 nm—pH-independent isobesticpoint) over that of the preincubation sample (zero release) normalizedto the increase in fluorescence obtained after lysis of preincubationsample with 0.2% Triton X-100 (100% release) (Kirpotin, D. et al., FEBSLetters, 388:115-118 (1996)). Results at various cysteine concentrationsfor liposome comprising the ortho-compound are shown in FIG. 8A and forthe for the para-compound are shown in FIG. 8B.

Example 8 In Vivo Characterization of Liposomes Comprising mPEG-DTB-DSPECompound

A. Liposome Preparation

The lipids partially hydrogenated phosphatidylcholine (PHPC),cholesterol and para-mPEG-DTB-DSPE (prepared as described in Example 1,mPEG MW=1980 Daltons) were dissolved in a 55:40:5 mole percent ratio,respectively, in an organic solvent. The solvent was removed by rotaryevaporation to form a dried lipid film. The film was hydrated withaqueous buffer containing diethylene triamine pentacetic acid (EDTA) toform liposomes. After downsizing the liposomes to an average diameter of120 nm unentrapped EDTA was removed and In¹¹¹ was added to the externalmedium. The liposomes were incubated for a time sufficient for In¹¹¹ tocross the lipid bilayer and chelate with EDTA.

B. In Vivo Administration

Mice were divided into two study groups. The liposome compositiondescribed above was injected into all test animals. One group of thetest animals also received a 200 μL injection of 200 mM cysteine at 1, 3and 5 hours post liposome injection. The other test group received aninjection of saline at the same time points. Liposome content in theblood was determined by monitoring blood samples for In¹¹¹. The resultsare shown in FIG. 10.

Although the invention has been described with respect to particularembodiments, it will be apparent to those skilled in the art thatvarious changes and modifications can be made without departing from theinvention.

1. A polypeptide composition having the general structure:

wherein R¹ is a hydrophilic polymer having a molecular weight of between440-100,000 Daltons and comprising a linkage for attachment to thedithiobenzyl moiety; R² is selected from the group consisting of H,alkyl, and aryl; R³ is selected from the group consisting of O(C═O),S(C═O), and O(C═S); R⁵ is selected from the group consisting of H, alkyland aryl; where the polypeptide is between 10-30 kDa and whereorientation of CH₂—R³ is selected from the ortho position and the paraposition; and n is at least
 1. 2. The composition of claim 1, whereinsaid hydrophilic polymer R¹ is selected from the group consisting ofpolyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline,polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropyl-methacrylamide, polymethacrylamide,polydimethyl-acrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide, copolymers thereof, andpolyethyleneoxide-polypropylene oxide.
 3. The composition of claim 1,wherein R¹ is polyethyleneglycol.
 4. The composition of claim 1, whereinR⁵ is H and R² is selected from the group consisting of H, OH₃, C₂H₅,and C₃H₈.
 5. The composition of claim 1, wherein R² and R⁵ are alkyls.6. The composition of claim 1, wherein n is between about 1 and about 6.7. The composition of claim 1, wherein the cytokine is a tumor necrosisfactor.
 8. A polypeptide composition, comprising: a polypeptideconjugated to at least one a hydrophilic polymer chain to form amodified polypeptide having the general structure:

wherein R¹ is a hydrophilic polymer having a molecular weight of between440-100,000 Daltons and comprising a linkage for attachment to thedithiobenzyl moiety; R² is selected from the group consisting of H,alkyl, and aryl; R³ is selected from the group consisting of O(C═O),S(C═O), and O(C═S); R⁵ is selected from the group consisting of H, alkyland aryl; where the polypeptide is between 10-30 kDa and whereorientation of CH₂—R³ is selected from the ortho position and the paraposition; and n is at least 1; and a pharmaceutically-acceptablecarrier.
 9. The composition of claim 8, wherein n is between about 1 andabout
 6. 10. The composition of claim 8, wherein said hydrophilicpolymer R¹ is selected from the group consisting ofpolyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline,polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropyl-methacrylamide, polymethacrylamide,polydimethyl-acrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide, copolymers thereof, andpolyethyleneoxide-polypropylene oxide.
 11. The composition of claim 8,wherein R¹ is polyethyleneglycol.
 12. The composition of claim 8,wherein R⁵ is H and R² is selected from the group consisting of H, CH₃,C₂H₅, and C₃H₈.
 13. The composition of claim 8, wherein R² and R⁵ arealkyls.
 14. The composition of claim 8, wherein the cytokine is a tumornecrosis factor.